Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Sampling
2.3. RNA Extraction, PCR Screening and Sequencing
2.4. Sequencing of Full-Length Genomes
2.5. Genome Analysis
2.6. Transcriptional Analysis of Subgenomic mRNA
2.7. Cell Lines, Gene Cloning, and Expression
2.8. Virus Isolation
2.9. Apoptosis Analysis
2.10. Dual Luciferase Reporter Assays
2.11. BtCoV/Rh/YN2012 Spike-Mediated Pseudoviruses Cell Tropism Screening
2.12. Nucleotide Sequence Accession Numbers
3. Results
3.1. CoVs Detected in Rhinolophus Bats
3.2. Genomic Characterization of a Novel Alpha-CoV (BtCoV/Rh/YN2012)
3.3. Subgenomic Structures and Accessory Genes of BtCoV/Rh/YN2012
3.4. Phylogenetic Analysis
3.5. Estimation of Synonymous and Nonsynonymous Substitution Rates
3.6. Apoptosis Analysis of ORF9
3.7. ORF4a Proteins Induce Production of IFN-β
3.8. ORF3a Proteins Modulate NF-κB
3.9. BtCoV/Rh/YN2012 Spike Mediated Pseudovirus Entry
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Bat Species | Samples | CoV Species Positive (Positive Rate %) | Province1 | ||||||
---|---|---|---|---|---|---|---|---|---|
HKU2 | HKU8 | HuB2013 | Mi-BtCoV 1 | SARSr-CoV | BtCoV/Rh/YN2012 | Other AlphaCoVs | |||
R.affinis | 499 | 52(10.4) | 2(0.4) | 2(0.4) | 3(0.6) | 5, 7, 8, 10, 13, 18, 19 | |||
R.blythi | 17 | 5,11,18 | |||||||
R.ferrumequinum | 238 | 1(0.4) | 12(5.0) | 1,3,4,7,9,10,14,15,18 | |||||
R.lepidus | 21 | 3 | |||||||
R.luctus | 8 | 5,8,19 | |||||||
R.macrotis | 31 | 1(3.2) | 1(3.2) | 2,5,6,9,11,13,1,18 | |||||
R.monoceros | 5 | 18 | |||||||
R.pearsoni | 106 | 3,5,6,7,10,17,18 | |||||||
R.pusillus | 283 | 4(1.4) | 2(0.7) | 3,5,6,9,10,11,13,14 | |||||
R.shamelli | 55 | 25(45.5) | 10(18.2) | 1(0.4) | 2(0.7)2 | 18 | |||
R.sinicus | 740 | 12(1.6) | 3(0.4) | 72(9.7) | 6(0.8) | 2(0.3)3 | 3,5,6,7,8,11,12,13,16,18,19 | ||
R.spp. | 58 | 2,6,7,18 | |||||||
Total | 2061 | 94(4.6) | 12(0.6) | 1(0.05) | 4(0.2) | 89(4.3) | 9(0.4) | 4(0.2) |
CoV | ORFs | Nucleotide Positions | Predicted Size (AA) of Protein | Pairwise AA Identity (%) | TRS Sequence | |||||
---|---|---|---|---|---|---|---|---|---|---|
RsYN1 | RsYN2 | RsYN3 | RaGD | HuB2013 | HKU10 | |||||
RsYN1 | 1ab | 298–20465 | 6722 | 93.0 | 93.0 | 83.7 | 81.8 | 76.4 | CTAAAC(216)ATG | |
S | 20472–24533 | 1353 | 68.4 | 67.9 | 64.9 | 62.3 | 61.7 | CTAAAT(8)ATG | ||
S1 | 20541–22634 | 698 | 60.2 | 59.4 | 56.7 | 53.7 | 53.2 | |||
S2 | 22659–24527 | 623 | 76.9 | 77.0 | 74.0 | 72.1 | 71.4 | |||
NS3a | 24533–25201 | 222 | 65.0 | 65.0 | 54.3 | 52.4 | 53.5 | CAATAC(26)ATG | ||
NS3b | 24978–25070 | 30 | NA | NA | NA | NA | NA | CCTTAC(35)ATG | ||
NS4a | 25222–25581 | 119 | 47.1 | 62.2 | 18.6 | NA | NA | CTTTAC(50)ATG | ||
NS4b | 25563–26009 | 148 | 57.8 | 52.9 | 11.4 | NA | NA | |||
E | 25993–26217 | 74 | 89.2 | 89.2 | 77.1 | 68.7 | 75.9 | CTAAAC(66)ATG | ||
M | 26229–26912 | 227 | 88.8 | 89.2 | 78.1 | 86.6 | 85.5 | CTAAAC(1)ATG | ||
N | 26923–28134 | 403 | 84.3 | 84.3 | 68.1 | 70.3 | 61.8 | CTAAAC(3)ATG | ||
NS8 | 28290–28409 | 39 | 26.7 | 33.3 | 28.6 | NA | NA | CTAAAC(0)ATG | ||
NS9 | 28454–28819 | 121 | 78.1 | 50.6 | 55.6 | NA | NA | TTTCAC(3)ATG | ||
Concatenated domains1 | 13,650 | 4550 | 97.3 | 97.4 | 91.9 | 84.2 | 86.9 | |||
RsYN2 | 1ab | 298–20435 | 6712 | 93.0 | 99.4 | 84.4 | 82.5 | 76.9 | CTAAAC(216)ATG | |
S | 20437–24495 | 1352 | 68.4 | 93.6 | 66.9 | 65.7 | 62.4 | CTAAAT(3)ATG | ||
S1 | 20509–22599 | 697 | 60.2 | 88.2 | 58.7 | 58.8 | 55.6 | |||
S2 | 22624–24489 | 623 | 76.9 | 99.9 | 76.3 | 72.8 | 70.0 | |||
NS3a | 24495–25154 | 219 | 65.0 | 100.0 | 55.9 | 52.8 | 55.5 | CGTTAC(26)ATG | ||
NS4a | 25164–25488 | 104 | 47.1 | 64.4 | 17.5 | NA | NA | CTTCAC(24)ATG | ||
NS4b | 25507–25953 | 148 | 57.8 | 94.2 | 12.5 | NA | NA | |||
E | 25934–26158 | 74 | 89.2 | 100.0 | 81.9 | 66.3 | 77.1 | CTAAAC(66)ATG | ||
M | 26170–26859 | 229 | 84.3 | 99.8 | 79.9 | 90.0 | 88.8 | CTAAAC(1)ATG | ||
N | 26870–28096 | 408 | 84.3 | 99.8 | 68.3 | 69.9 | 63.0 | CTAAAC(3)ATG | ||
NS8 | 28107–28385 | 92 | 26.7 | 66.7 | 64.8 | NA | NA | CTAAAC(0)ATG | ||
NS9 | 28425–28790 | 121 | 78.1 | 77.5 | 58.1 | NA | NA | TTTCAC(3)ATG | ||
Concatenated domains | 13641 | 4547 | 97.3 | 99.6 | 91.8 | 84.3 | 87.1 | |||
RsYN3 | 1ab | 298–20435 | 6712 | 93.0 | 99.4 | 84.4 | 82.5 | 76.9 | CTAAAC(216)ATG | |
S | 20437–24495 | 1352 | 67.9 | 93.6 | 66.6 | 65.8 | 62.4 | CTAAAT(3)ATG | ||
S1 | 20506–22599 | 698 | 59.4 | 88.2 | 57.9 | 59.2 | 54.9 | |||
S2 | 22621–24489 | 623 | 77.0 | 99.9 | 76.4 | 72.9 | 70.1 | |||
NS3a | 24495–25154 | 219 | 65.0 | 100.0 | 55.9 | 52.8 | 55.5 | CGTTAC(26)ATG | ||
NS4a | 25164–25526 | 120 | 62.2 | 64.4 | 17.6 | NA | NA | CTTCAC(24)ATG | ||
NS4b | 25508–25954 | 148 | 52.9 | 94.2 | 12.5 | NA | NA | |||
E | 25935–26159 | 74 | 89.2 | 100.0 | 81.9 | 66.3 | 77.1 | CTAAAC(66)ATG | ||
M | 26171–26860 | 229 | 89.2 | 99.6 | 80.3 | 90.3 | 89.2 | CTAAAC(1)ATG | ||
N | 26871–28097 | 408 | 84.3 | 99.8 | 68.3 | 69.9 | 62.8 | CTAAAC(3)ATG | ||
NS8 | 28108–28386 | 92 | 33.3 | 66.7 | 63.8 | NA | NA | CTAAAC(0)ATG | ||
NS9 | 28434–28793 | 119 | 71.2 | 77.5 | 50.6 | NA | NA | TTTCAC(3)ATG | ||
Concatenated domains | 13641 | 4547 | 97.4 | 99.6 | 91.8 | 84.4 | 87.2 | |||
RaGD | 1ab | 296–20466 | 6723 | 83.7 | 84.4 | 84.4 | 80.0 | 76.4 | CTAAAC(215)ATG | |
S | 20463–24542 | 1359 | 64.9 | 66.9 | 66.6 | 61.9 | 62.4 | CTAAAC(4)ATG | ||
S1 | 20520–22652 | 711 | 56.7 | 58.7 | 57.9 | 53.6 | 54.5 | |||
S2 | 22674–24535 | 621 | 74.0 | 76.3 | 76.4 | 71.4 | 72.2 | |||
NS3a | 24542–25198 | 218 | 54.3 | 55.9 | 55.9 | 48.0 | 52.0 | CGTTAC(26)ATG | ||
NS4a | 25200–25663 | 157 | 18.6 | 17.5 | 17.6 | NA | NA | CTTTGC(34)ATG | ||
NS4b | 25294–25611 | 105 | 11.4 | 12.5 | 12.5 | NA | NA | |||
E | 25654–25878 | 74 | 77.1 | 81.9 | 81.9 | 63.9 | 81.9 | CCAAAC(66)ATG | ||
M | 25884–26624 | 246 | 78.1 | 79.9 | 80.3 | 78.1 | 78.8 | CTAAAC(3)ATG | ||
N | 26635–27810 | 391 | 68.1 | 68.3 | 68.3 | 67.3 | 60.8 | CTAAAC(3)ATG | ||
NS8 | 27821–28099 | 92 | 28.6 | 64.8 | 63.8 | NA | NA | CTAAAC(0)ATG | ||
NS9 | 28123–28485 | 120 | 55.6 | 58.1 | 50.6 | NA | NA | CTTTAC(3)ATG | ||
Concatenated domains | 13,674 | 4558 | 91.9 | 91.8 | 91.8 | 83.0 | 87.4 |
NSP | Putative Functional Domain(s) | RsYN1 | RsYN2 | RsYN3 | RaGD | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Amino Acides Position in ORF1ab | Predicted Size (aa of Protein) | C-end Predicted Cleavage Site | Amino Acides Position in ORF1ab | Predicted Size (aa of Protein) | C-End Predicted Cleavage Site | Amino Acides Position in ORF1ab | Predicted Size (aa of Protein) | C-end Predicted Cleavage Site | Amino Acides Position in ORF1ab | Predicted Size (aa of Protein) | C-end Predicted Cleavage Site | ||
NSP1 | Unknown | M1–A195 | 195 | VA│AP | M1-A195 | 195 | VA│SP | M1-A195 | 195 | VA│SP | M1-A195 | 195 | TA│PP |
NSP2 | Unknown | A196–C896 | 701 | RC│GG | S196-S889 | 694 | RC│GG | S196-S889 | 694 | RC│GG | P196-S889 | 694 | RS│GG |
NSP3 | ADRP, PL2 pro | G897–G2463 | 1567 | QG│SG | G890-G2453 | 1564 | AG│SG | G890-G2453 | 1564 | AG│SG | G890-G2462 | 1573 | NG│SG |
NSP4 | Hydrophobid domain | S2464–Q2941 | 478 | LQ│SG | S2454–Q2931 | 478 | LQ│SG | S2454-Q2931 | 478 | LQ│SG | S2463-Q2940 | 478 | LQ│SG |
NSP5 | 3CL pro | S2942–Q3243 | 302 | LQ│ST | S2932–Q3233 | 302 | LQ│ST | S2932-Q3233 | 302 | LQ│ST | S2941-Q3242 | 302 | LQ│SN |
NSP6 | Hydrophobid domain | S3244–Q3519 | 276 | VQ│SK | S3234–Q3509 | 276 | VQ│SK | S3234-Q3509 | 276 | VQ│SK | S3243-Q3518 | 276 | VQ│SK |
NSP7 | Replicase | S3520–Q3602 | 83 | LQ│SV | S3510–Q3592 | 83 | LQ│SV | S3510-Q3592 | 83 | LQ│SV | S3519-Q3601 | 83 | LQ│SV |
NSP8 | Replicase | S3603–Q3797 | 195 | LQ│NN | S3593–Q3787 | 195 | LQ│NN | S3593-Q3787 | 195 | LQ│NN | S3602-Q3796 | 195 | LQ│NN |
NSP9 | Replicase | N3798–Q3905 | 108 | LQ│AG | N3788–Q3895 | 108 | LQ│AG | N3788-Q3895 | 108 | LQ│AG | N3797-Q3904 | 108 | LQ│AG |
NSP10 | RNA synthesis protein | A3906–Q4041 | 136 | VQ│SL | A3896–Q4031 | 136 | VQ│AL | A3896-Q4031 | 136 | VQ│AL | A3905-Q4040 | 136 | VQ│SL |
NSP11 | Unknown (short peptide at the end of ORF1a) | S–D | 17 | A-D | 17 | A-D | 17 | S-N | 17 | ||||
NSP12 | RdRp | S4042–Q4968 | 927 | LQ│AA | A4032-Q4958 | 927 | LQ│AA | A4032-Q4958 | 927 | LQ│AA | S4041-Q4967 | 927 | LQ│AA |
NSP13 | Hel, NTPase | A4969–Q5565 | 597 | LQ│AG | A4959-Q5555 | 597 | LQ│AG | A4959-Q5555 | 597 | LQ│AG | A4968-Q5564 | 597 | LQ│AG |
NSP14 | ExoN, NMT | A5566–Q6083 | 518 | LQ│GL | A5556-Q6073 | 518 | LQ│GL | A5556-Q6073 | 518 | LQ│GL | A5565-Q6082 | 518 | LQ│GL |
NSP15 | NeudoU | G6084–Q6422 | 339 | LQ│AG | G6074-Q6412 | 339 | LQ│SG | G6074-Q6412 | 339 | LQ│SG | G6083-Q6421 | 339 | LQ│SG |
NSP16 | 2’-O-MT | A6423–K6722 | 300 | S6413-K6712 | 300 | S6413-K6712 | 300 | S6422-K6723 | 302 |
Gene | Ka/Ks Ratio |
---|---|
NSP1 | 0.256 |
NSP2 | 0.390 |
NSP3 | 0.315 |
NSP4 | 0.276 |
NSP5 | 0.128 |
NSP6 | 0.323 |
NSP7 | 0.067 |
NSP8 | 0.125 |
NSP9 | 0.230 |
NSP10 | 0.098 |
NSP11 | 0.236 |
NSP12 | 0.073 |
NSP13 | 0.045 |
NSP14 | 0.121 |
NSP15 | 0.124 |
NSP16 | 0.083 |
S | 0.258 |
ORF3a | 0.357 |
ORF4a | 0.727 |
ORF4b | 0.623 |
E | 0.062 |
M | 0.0691 |
N | 0.230 |
ORF8 | 0.276 |
ORF9 | 0.843 |
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Wang, N.; Luo, C.; Liu, H.; Yang, X.; Hu, B.; Zhang, W.; Li, B.; Zhu, Y.; Zhu, G.; Shen, X.; et al. Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats. Viruses 2019, 11, 379. https://doi.org/10.3390/v11040379
Wang N, Luo C, Liu H, Yang X, Hu B, Zhang W, Li B, Zhu Y, Zhu G, Shen X, et al. Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats. Viruses. 2019; 11(4):379. https://doi.org/10.3390/v11040379
Chicago/Turabian StyleWang, Ning, Chuming Luo, Haizhou Liu, Xinglou Yang, Ben Hu, Wei Zhang, Bei Li, Yan Zhu, Guangjian Zhu, Xurui Shen, and et al. 2019. "Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats" Viruses 11, no. 4: 379. https://doi.org/10.3390/v11040379
APA StyleWang, N., Luo, C., Liu, H., Yang, X., Hu, B., Zhang, W., Li, B., Zhu, Y., Zhu, G., Shen, X., Peng, C., & Shi, Z. (2019). Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats. Viruses, 11(4), 379. https://doi.org/10.3390/v11040379